Epoxidation of cis-propenylphosphonic acid

ABSTRACT

Cis-propenylphosphonic acid and its salts are converted to ()(cis-1,2-epoxypropyl)phosphonic acid by intimately contacting said acid or salts with epoxidizing enzymes of molds. The epoxyphosphonic acid and its derivatives are valuable antibiotics active against gram-positive and gram-negative bacteria.

llnite States Patent White 1 3,657,67 1451 Apr. 18,1972

[ IEPOXIDATION OF CIS- PROPENYLPHOSPHONIC ACID [72] Inventor: Raymond F.White, Englishtown, NJ.

[73] Assignee: Merck & Co., Inc., Rahway, NJ.

[22] Filed: Nov. 5, 1969 [21] Appl. No.: 874,387

[52] US. Cl. 51 Int. Cl. [58] Field of Search ..195/81, 195/51 R ..Cl2d9/00 ....l95/51 R, 28 R, 81

[56] References Cited UNITED STATES PATENTS 4/1958 Shull et a1 ..195/51OTHER PUBLICATIONS Microbes as Chemical Reagents, Chemical and Engineer-Primary Examiner-Joseph M. Golian Att0rney-John Frederick Gerkens, .1.Jerome Behan and 1. Louis Wolk [57] ABSTRACT Cis-propenylphosphonic acidand its salts are converted to ()(cis-1,Z-epoxypropyl)phosphonic acid byintimately contacting said acid or salts with epoxidizing enzymes ofmolds. The epoxyphosphonic acid and its derivatives are valuableantibiotics active against gram-positive and gram-negative bacteria.

10 Claims, N0 Drawings ll EPOXIDATION OF CIS-PROPENYLPHOSPHONIC ACIDBACKGROUND OF THE INVENTION Processes for preparing (cis-l,2-epoxypropyl) phosphonic acid and salts thereof have been described inthe art. Thus, this phosphonic acid has been obtained by growing certainspecies of Streptomyces, such as Streptomycesfradt'ae, in suitablefermentation media, and it has also been prepared chemical synthesis ofthe racemic acid and resolution of the racemic mixture. Both of thesemethods of producing the antibiotic substance possess disadvantages; theyields of the fermentation process being low and the resolution step inthe synthetic process being difficult to carry out on a commercialscale. Accordingly, other methods of preparing the antibiotic substanceand salts thereof have been sought which would avoid the difficulties ofthe known processes.

SUMMARY OF THE INVENTION It is an object of this invention to provide animproved method of converting cis-propenylphosphonic acid and its saltsto (cis-l ,2-epoxypropyl)phosphonic acid and its salts in good yieldwithout the concomitant production of the unwanted isomer. Anotherobject is to provide a method of epoxidizing cis-propenylphosphonic acidand its salts in aqueous solution from which the desired epoxyphosphonicacid can be readily recovered. Other objects will be apparent from thedetailed description of the invention hereinafter provided.

In accordance with this invention, cis-propenyl-phosphonic acid or asalt thereof is intimately contacted with epoxidizing enzymes ofmicroorganisms to produce (cis-l,2-epoxypropyl)phosphonic acid and saltsthereof. The biochemical epoxidation of this invention is brought aboutwith the epoxidizing activity produced by suitable species of fungi,preferably by a species of mold such as various species of Fungiimperfecti. These molds can be obtained from culture collections orisolated from natural sources such as soil in accordance with methodsand techniques well known in this art. Examples of suitable molds thatmight be mentioned are various species of Penicillium, such as P.purpurrescens, P. aculeatum, P. crustosum, P. charesii, P. corylophilum,P. sopii, P. puberulum, P. multicolor, P. funiculosum, P. spinulosum, P.frequentans, P. palitans, P. purpurogenum, and P. gilmanii, variousspecies of Paecilomyces such as P. varioti, and various species ofOidium.

The suitability of specific molds for effecting the epoxidation can bereadily determined by growing a mold in suitable media, such as thoseshown in the examples, containing 2507/ml. of a cis-propenylphosphonicacid salt until good growth of the culture is obtained (6 to 10 days),and then testing the culture broth for biological activity by assay withsensitive and resistant strains of Proteus vulgaris. The cultures havingactivity against the sensitive strain and no activity against theresistant strain have the desirable epoxidizing activity. The assayswith Proteus vulgaris can be carried out by the disc assay proceduresdescribed in the art using Proteus vulgaris NRRL-B336l as the sensitivestrain and with Proteus vulgaris NRRL-B3722 as the(cis-l,2-epoxypropyl)phosphonate resistant strain.

The process of this invention is carried out by intimately contactingthe cis-propenylphosphonic acid compound with the epoxidizing enzyme ofthe mold. This can be done by culturing the mold in a suitable nutrientmedium for growing the mold and adding the phosphonate at the beginningof the fermentation period. Alternatively, the phosphonate canbe addedaeseptically after the mold has been growing for some time.

Pursuant to another specific embodiment of this invention, theepoxidation can be effected by contacting the phosphonate compound withthe enzymes produced by growing suitable molds. Thus, the epoxidation iseffected by contacting the resting cells of the mold in an aqueousmedium, or by first separating the enzymes from the cells in accordancewith methods known in this art and contacting the phosphonate with theseparated enzymes in a suitable aqueous medium. These latter methodshave the advantage of simplifying the recovery of the(cis-l,2-epoxypropyl)phosphonic acid salt, although it is generallypreferred to add the propenyl compound to the medium in which the moldis grown and permitting the growth or fermentation to continue in thepresence of the cis-propenylphosphonate.

The aqueous nutrient media suitable for growing the epoxidizing strainsshould contain sources of carbon and nitrogen assimilable by the mold aswell as minor amounts of inorganic salts necessary for its growth.Sources of assimilable carbon usually used in fermentation processessuch as dextrose, invert molasses, beet molasses, cerelose, and the likecan be used. Similarly, sources of nitrogen employed in fermentationprocesses such as peptones, soybean meal, corn steep liquor,polypeptides, enzymatic digests of casein and the like can be utilized.In addition, the medium can contain minor amounts of mineralconstituents such as soluble salts of magnesium, sodium, potassium, andthe like as well as growth factors, such as vitamins, or other growthstimulating substances can be added to the medium. Suitable media areshown in the illustrative examples hereof.

The nutrient medium is usually inoculated with a vegetative growth ofthe mold although it can also be inoculated by ad ding spores of themold to the medium. Growth of the microorganism is promoted byconducting the incubation at temperatures of between about roomtemperature and 30 C. although higher or lower temperatures can be useddepending upon the optimal temperature for growing particular molds. ThepH of the medium is not critical and is adjusted to that required foroptimal growth of the particular mold, usually between about 6.0 and8.0. The growth of the mold is carried out aerobically in accordancewith methods well known in this art. The time required to effect thedesired epoxidation varies with the particular mold used, but generallyit is desirable to permit the fermentation to continue until good growthof the mold is achieved. Thus, it may be necessary to continue thefermentation for as long as 10 days.

.The concentration of the cis-propenylphosphonate in the medium is notunduly critical and concentrations of 0.1 to 5 g. per liter can be used;the optimum amount for each mold being readily determined by a fewsimple experiments. The phosphonate salt can be added to the mediumcontaining the epoxidizing enzymes, or one in which they are produced,in the form of a solution in a suitable biologically acceptable solvent;for example, water, a lower alcohol or a glycol, in the form of asuspension in a suitable liquid, or in the form of a solid, preferablyin a very finely divided form. Generally, it is preferred to add thephosphonate to the medium in the form of a water solution of a solublesalt such as an alkali metal salt or an amine salt, although it can alsobe added as an insoluble salt or as the acid per se.

The processes of this invention produce primarily the form of (cis-l,2-epoxypropyl)phosphonic acid which can then be readily recovered fromthe medium in which the epoxidation is carried out by methods known inthis art. Thus, it can be adsorbed on a basic anion exchange resin onthe chloride cycle and then eluted with aqueous sodium chloride.Alternatively, the epoxyphosphonic acid can be separated by adsorptionon activated alumina from which it can be eluted with aqueous or aqueousalcoholic solutions of ammonium hydroxide.

The examples which follow illustrate methods of carrying out the presentinvention.

EXAMPLE I A medium containing 0.8 percent nutrient broth (Difco), 0.2percent yeast extract, 3 percent dextrose and 0.3 percent malt extractin distilled water was prepared and the pH adjusted to 7.0. 10 ml. ofthe resulting medium was dispensed to 25 X 200 mm. test tubes which werethen sterilized by'autoclaving at 121 C. for 15 minutes.

An inoculum suspension was prepared by adding a lyophilized culture ofPenicillium purpurrescens NRRL-72O to ml. of the sterilized mediumdescribed above. The resulting inoculum suspension was used to inoculateone of the test tubes containing ml. of the sterilized medium and asecond tube containing 10 ml. of the sterilized medium to which wasadded 1 ml. of a sterile aqueous solution containing 2507 of sodiumcis-propenylphosphonate.

The inoculated tubes were incubated at 28 C. on a mechanical shakeroperating at 220 rpm until good growth of the culture was observed.After 6 days the incubated broths were centrifuged (25,000 X G) toseparate the cells and the supernatant broth was examined for biologicalactivity by disc assay with sensitive and resistant strains of Proteusvulgaris with the following results:

Inhibition Zone (m Sensitive Resistant Control medium 0 0 Mediumcontaining phosphonate 21 0 An uninoculated tube containing 10 ml. ofthe sterilized medium and 1 ml. of an aqueous solution containing 2507'of sodium cis-propenylphosphonic acid was also incubated with the twoinoculated tubes. After 6 days the tube was centrifuged and thesupernatant liquid was found to be inactive in the Proteus vulgarisassays.

EXAMPLES 2-20 Using the procedures of Example 1 with the molds listedbelow, the following results were obtained with the inoculated tubescontaining 2507 of sodium cis-propenyl-phosphonate:

Inhibition Zone Sensi- Resist- Ex. No. Mold tive ant 2 P. cruxmrumNRRL-949 24.0 0 3 P. charesii NRRL-778 35.0 0 4 P. aculearum QM-284031.5 0 5 P. curylophilum Qm-3l l5 25.5 0 6 Paerilomyce: variari 27.0 0 7Oidium sp. Qm-696 26.0 0 8 P. .wppr'i NRRL-9l2 42.0 0 9 P.funicul0.rumNRRL-l I32 23.0 0 l0 P. puberulum NRRL-988 16.0 0 l l P. multicolorQm-96d 26.0 0 l2 P. funiculosum NRRL-l768 33.0 0 l3 P. frequenranrNRRL-l 189 20.0 0 l4 P. rpinulosum NRRL-727 32.0 0 [5 P. :pinulasumNRRL-728 41.0 0 l6 P. :pinulusum QM-l08b 37.0 0 l7 P. frequenlan:NRRL-763 34.0 0 18 P. palirans NRRL-966 32.0 0 I) P. purpurogenum QM-l7jI50 0 20 P. gilmrmif 26.0 0

l. lnstituo Oswaldo Cruz, Rio de Janicro, Brazil 2. Dr. F. Rice,American University, Washington, DC.

NRRL- Northern Utilization Research and Development Division, U. S.Departmcnt ofAgriculture, Peoria, Illinois QM Army QuatermastcrCollection, Quatermaster Research and Development Center, U. S. Army,Natick, Massachusetts In each instance the clarified broth of thecontrol test run in the same medium without the added phosphonate salthad no activity when tested by the same assay procedures.

EXAMPLE 21 A medium consisting of 0.8 percent nutrient broth, 0.2percent yeast extract, 3 percent cerelose and 0.3 percent malt extractwas prepared and the pH adjusted to 7.0. The medium was dispensed, 40ml. to a 250 ml. Erlenmeyer flask, autoclaved at 121 C., p.s.i. for 15minutes. The medium was inoculated with a loopful of inoculum from anagar slant with one of the following microorganisms: Penicilliumpurpurrescens NRRL-720, P. soppii NRRL-9l2, P. spinulosum NRRL-728.

The flasks were incubated on a mechanical shaker (220 rpm) at 28 C.until the cultures were well grown (2 to 4 days). This inoculumsuspension was used to inoculate flasks containing 40 ml. of the mediumdescribed above and flasks containing 40 ml. of the medium to whichcis-propenylphosphonic acid had been added (200y/ml.). The flasks wereincubated on a mechanical shaker (220 rpm) at 28 C. until the cultureswere well grown (6 to 10 days). After incubation the cells were removedby centrifugation (25,000 X G) and the supernatant fluids examined forbiological activity by disc assay with sensitive and resistant strainsof Proteus vulgaris.

The activity of 450 ml. of fermentation broth, obtained by growingPenicillium soppii NRRL-9l2 in shake flasks as described above, wasadsorbed on a 36 cm. column of a strongly basic polystyrene type anionexchange resin (Dowex l X 2) on the chloride cycle. The resin adsorbatewas then eluted with 3 percent aqueous sodium chloride in fractions of 5ml. each. Fractions 15 through 24 containing all of the bioactivity werecombined and freeze dried to afford 1.94 g. of solids. The solids weredissolved in 10.0 ml. of water and adjusted to pH 5.70 by the additionof 1.65 ml. of 2.5N I-ICl. This solution was passed through a 15 cm.column of acid washed alumina and then washed with 15 ml. of water,followed by 15 ml. of 3:1 methanol-water. All of the bio-activity wasadsorbed on the column of alumina and was eluted with 1N ammoniumhydroxide in 3:1 methanol-water in fractions of 5 ml. each. Thebio-activity, which started to appear in fraction 3, was present infractions 3 to 8; most of the activity being present in fractions 3 and4. Fraction 3, which contained 3.00 mg. of(cis-1,2-epoxypropyl)phosphonic acid by bioassay, was freeze dried toafford 324.0 mg. of solid residue. This solid product was slurried threetimes with 3 ml. of methanol with slight warming and the extracts wereconcentrated to obtain 120.6 mg. of amorphous solids which by bioassaycontained 1.85 percent of (cis-l,2-epoxypropyl)phosphonic acid. Vaporphase chromatography of the amorphous solids showed that the solidscontained 1.2 percent of (cis-l,2-epoxypropyl)phosphonic acid and noneof the isomer.

Various changes and modifications in the procedures herein disclosedwill occur to those skilled in the art, and to the extent that suchchanges and modifications are embraced by the appended claims, it is tobe understood that they constitute part of our invention.

Iclaim:

l. A process for producing (cis-1,2-epoxypropyl)phosphonic acid or asalt thereof which comprises intimately contactingcis-propenylphosphonic acid or a salt thereof in an aqueous medium withan epoxidizing enzyme produced by growing a mold of the class of Fungiimperfecti in an aqueous nutrient medium.

2. The process of claim 1 wherein the mold is a species of Penicillium.

3. The process of claim 1 wherein the mold is P. purpurrescens, P.crustosum, P. charesii, P. aculeatum, P. corylophilum, Paecilomycesvarioti, Oidium sp., P. soppii, P. funicu- Iosum, P. puberulum, P.multicolor, P. spinulosum, P. frequentans, P. palitans, P. purpurogenumor P. gilmanii.

4. A process for producing (cis-1,2-epoxypropyl)phosphonic acid or asalt thereof which comprises growing an epoxidizing mold of the class ofFungi imperfecti in an aqueous nutrient medium in the presence ofcis-prope nylphosphonic acid or a salt thereof.

5. The process of claim 4 wherein the mold is a species of Penicillium.

6. The process of claim 4 wherein the mold is P. purpurrescens, P.crustosum, P. charesii, P. aculeatum, P. corylophilum, Paecilomycesvarioti, Oidium sp., P. soppii, P. funiculosum, P. puberulum, P.multicolor, P. spinulosum, P. frequentans, P. palitans, P. purpurogenumor P. gilmanii.

A process for producing (cisl,2-epoxypropyl)phosphonic acid or a saltthereof which comprises intimately contacting cis-propenylphosphonicacid or a salt thereof in an aqueous medium with an epoxidizing enzymeproduced by growing a mold of the class of Fungi imperfecti in anaqueous nutrient medium and recovering (cis-l,2-epoxypropyl)phosphonicacid or a salt thereof from the resulting reaction mixture.

8. The process of claim 7 wherein the mold is a species of Penicillium.

9. The process according to claim 7 wherein the mold is grown in thepresence of cis-propenylphosphonic acid or a salt thereof.

10. The process of claim 7 wherein the mold is P. purpur' rescens, P.crustosum, P. charesii, P. aculeatum, P. corylophilum, Paecilomycesvarioti, Oidium sp., P. soppii, P. funiculosum, P. puberulum, P.multicolor, P. spinulosum, P. frequentans, P. palitans, P. purpurogenumor P. gilmanii.

2. The process of claim 1 wherein the mold is a species of Penicillium.3. The process of claim 1 wherein the mold is P. purpurrescens, P.crustosum, P. charesii, P. aculeatum, P. corylophilum, Paecilomycesvarioti, Oidium sp., P. soppii, P. funiculosum, P. puberulum, P.multicolor, P. spinulosum, P. frequentans, P. palitans, P. purpurogenumor P. gilmanii.
 4. A process for producing (-)(cis-1,2-epoxypropyl)phosphonic acid or a salt thereof which comprisesgrowing an epoxidizing mold of the class of Fungi imperfecti in anaqueous nutrient medium in the presence of cis-propenylphosphonic acidor a salt thereof.
 5. The process of claim 4 wherein the mold is aspecies of Penicillium.
 6. The process of claim 4 wherein the mold is P.purpurrescens, P. crustosum, P. charesii, P. aculeatum, P. corylophilum,Paecilomyces varioti, Oidium sp., P. soppii, P. funiculosum, P.puberulum, P. multicolor, P. spinulosum, P. frequentans, P. palitans, P.purpurogenum or P. gilmanii.
 7. A process for producing (-)(cis-1,2-epoxy-propyl)phosphonic acid or a salt thereof which comprisesintimately contacting cis-propenylphosphonic acid or a salt thereof inan aqueous medium with an epoxidizing enzyme produced by growing a moldof the class of Fungi imperfecti in an aqueous nutrient medium andrecovering (cis-1,2-epoxypropyl)phosphonic acid or a salt theReof fromthe resulting reaction mixture.
 8. The process of claim 7 wherein themold is a species of Penicillium.
 9. The process according to claim 7wherein the mold is grown in the presence of cis-propenylphosphonic acidor a salt thereof.
 10. The process of claim 7 wherein the mold is P.purpurrescens, P. crustosum, P. charesii, P. aculeatum, P. corylophilum,Paecilomyces varioti, Oidium sp., P. soppii, P. funiculosum, P.puberulum, P. multicolor, P. spinulosum, P. frequentans, P. palitans, P.purpurogenum or P. gilmanii.